Proteomics offers a more direct look at the biological functions of a cell or organism than does genomics, the traditional focus for evaluation of gene activity. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantitating expression at the protein level, rather than at the messenger RNA level. Proteomics also involves the study of non-genome encoded events including the post-translational modification of proteins, protein degradation and protein byproducts, interactions between proteins, and the location of proteins within the cell. The structure, function, or level of activity of the proteins expressed by a cell are also of interest.
The study of gene expression at the protein level is important because many of the most important cellular processes are regulated by the protein status of the cell, not by the status of gene expression. Also, the protein content of a cell is highly relevant to drug discovery efforts since most drugs are designed to be active against protein targets.
Current technologies for the analysis of protein mixtures, such as the intracellular proteins of a cell or population of cells and the proteins secreted by the cell or population of cells or biological fluids, are based on a variety of protein separation techniques followed by identification and/or analysis of the separated proteins. The most popular method is based on 2D-gel electrophoresis followed by “in-gel” proteolytic digestion and mass spectroscopy. Alternatively, Edman degradation and related methods may be used for the sequencing. This 2D-gel technique requires large sample sizes, is time consuming, and is currently limited in its ability to reproducibly resolve a significant fraction of the proteins expressed by a human cell. Techniques involving some large-format 2D-gels can produce gels which separate a larger number of proteins than traditional 2D-gel techniques, but reproducibility is still poor and over 95% of the spots cannot be sequenced due to limitations with respect to sensitivity of the available sequencing techniques. The electrophoretic techniques are also plagued by a bias towards proteins of high abundance.
Thus, there is a need for the ability to assay more completely proteins expressed by a cell or a population of cells in an organism or in a fluid comprising protein (such as serum, plasma, lymph, and other biological fluids), including up to the total set of proteins expressed by the cell or cells or found in the fluid comprising protein.
Many currently available methods for protein profiling have severe limitations with respect to sensitivity and throughput. There is a need for improved methods for simultaneously characterizing the large number of proteins found in biological samples.